In a fluorescence detecting apparatus, for example, an evanescent field is created on the surface of the substrate by the total internal reflection of light in the optically transparent substrate and biological molecules marked with fluorescence by a probe in the liquid sample supplied on the surface of the substrate are excited in the evanescent field. And the fluorescence radiated from the biological molecules as a result thereof are detected to detect qualitatively biological molecules or to analyze qualitatively the same.
Regarding such a detection of fluorescence, Funatsu et al., Nature vol. 374, 555-559 (1995) describes an apparatus that irradiates a prism with a laser beam and causes the total internal reflection of the laser beam to create an evanescent field in the sample solution on the prism, collects and detects the fluorescence radiating from the biological molecules within the sample excited by the evanescent field with an objective lens. Since an evanescent field is localized on the surface of a prism, the region where fluorescence or scattered light is excited in the sample solution is limited to the vicinity of the surface of the prism. As a result, the background light is contained low, and the molecules near the surface can be detected with a very high sensitivity. As a matter of fact, Funatsu et al., Nature vol. 374, 555-559 (1995) describes the success of detecting a single fluorescent molecule.
And JP-T No. 2004-527741 describes an apparatus that detects emission of light from molecules marked by fluorescence by exciting an evanescent field by having the excited beam totally reflected on the surface of a transparent body.
Generally in order to obtain a total internal reflection, beams must be irradiated obliquely on the reflecting surface. FIG. 1 shows a cross sectional view along the plane of incidence of how the excited beam 3 is irradiated from the inside of the substrate 1 having optical transparency into the boundary face between the substrate 1 and the sample 2 contiguous thereto to be totally reflected thereby. Here, the term “plane of incidence” means a surface drawn along the optical axis of the incident beam and the normal of the boundary surface, and the angle formed by the normal of the boundary surface and the optical beam of the incident beam is called “incident angle.” If the refractive index of the substrate 1 is represented by n1, the refractive index of the sample by n2 and the incident angle by θ, the necessary and sufficient condition for inducing a total internal reflection is shown by the following equation.sin θ>(n2/n1)  (Equation 1)
If this condition for a total internal reflection is satisfied, an evanescent field 5 is created only near the boundary surface in the sample. Incidentally, since in the case of a transparent material in the visible range, n1<2.5, and in the case of a aqueous solution sample, n2>1.3, always (n2/n1)>0.5. Therefore, if the equation 1 is satisfied, θ>30°. Since with ordinary glass n1 to 1.5, in most cases θ≧60°. Actually, in Funatsu et al., Nature vol. 374, 555-559 (1995), θ=68°.
And in Funatsu et al., Nature vol. 374, 555-559 (1995), the beam outputted by gas laser, having passed through the lens, is irradiated obliquely. Generally, the cross section of the beam outputted by gas laser is circular, and even if a beam having a circular section is made to pass through a lens, its section remains circular. When a beam having a circular cross section is irradiated on a reflection surface with an incident angle θ, the irradiated region on the reflection surface turns into an ellipse with an aspect ratio of cos θ. Therefore, the region irradiated with an excited beam in the apparatus described in Funatsu et al., Nature vol. 374, 555-559 (1995) is an ellipse with an aspect ratio of cos 68°=0.37. In Funatsu et al., Nature vol. 374, 555-559 (1995), a quartz substrate with n1=1.46 is used. As described above, even if whatever material may be used, the total internal reflection occurs always when θ>30° and in most cases θ>60°. Therefore, as far as the same configuration as Funatsu et al., Nature vol. 374, 555-559 (1995) is used, whatever substrate material may be used, the irradiated region always turns out to be an ellipse with an aspect ratio of less than cos 30°=0.86, and in most cases less than cos 60°=0.5.